Gastrointestinal Stromal Tumor (GIST) is the most common sarcoma with ~6,000 new cases annually in the U.S. GIST therapy is predicated upon KIT oncogene-addiction which drives sarcomagenesis. As a result, the ATP-dependent (-mimetic) KIT inhibitor, imatinib, is the first-line treatment for advanced GIST. Despite imatinib therapy, disease recurrence/progression remain frequent because GISTs develop imatinib-resistance caused by secondary KIT mutations. These mutations decrease the efficacy of other ATP-mimetics. Thus, compounds that target kinases, while avoiding ATP-mimetic resistance mechanisms, are of distinct interest. My mentor, David Cheresh, has reported the development of allosteric kinase inhibitors (AKI) that prevent KIT/BRAF/CRAF/PDGFR kinase activation in an ATP-independent manner. While current ATP-mimetics hit the right target(s), AKIs may: 1) have higher specificity than ATP-mimetics; 2) avoid gatekeeper mutations which render ATP-mimetics inactive; and 3) produce strong activity against imatinib-resistant GIST with fewer concerns for AKI-resistance. We hypothesize that this novel class of AKIs with KIT inhibition may be used to treat imatinib-resistant GIST, a disease for which there are limited therapeutic options. We aim to define the functional/mechanistic roles of allosteric kinase inhibition within the context of imatinib-resistant GIST. In Aim 1, we will characterize a series of highly potent AKIs using imatinib-sensitive and -resistant GIST cell lines. We will define AKI effects on cell viability, apoptosis, and cell cycle arrest in a time-/dose-dependent fashion. In Aim 2, we will perform biochemical/mechanistic analyses to determine the kinetic effects and binding affinities of lead AKIs on wild-type versus mutant KIT enzymes. In Aim 3, we will employ genetic and pharmacologic analyses to investigate how KIT (and BRAF/CRAF/PDGFR) AKIs influence intracellular kinase signaling pathways, which control fundamental processes such as tumor cell proliferation, survival, apoptosis and necrosis in imatinib-resistant lines. [We will also define AI effects upon KIT adapter protein interactions.] In Aim 4, we will investigate the lead AKIs identified in Aims 1-3 for inhibition of GIST growth using tumor xenograft/transgenic mouse models according to our IACUC-approved protocol. We will study novel compounds that target KIT via a distinct mechanism both in vitro and in vivo in order to target imatinib- resistant GIST and identify innovative anti-tumor agent(s). These studies will serve as the basis for clinical examination of AKIs directed against these validated kinase targets in patients with imatinib-resistant GIST. If efficacious agents are identified in vitro and in vivo, we intend to pursue additional funding to support moving these agent(s) to the bedside in the form of clinical trials